The Effects Of Total Glucosides Of Paeony On Mouse T Cells Proliferation And Activation Induced Cell Death In Vitro

Aim:To investigate the effects of total glucosides of paeony(TGP)on mouse T cells proliferation or activation induced cell death in vitro and the mechanism of immunosuppressive effects.Methods:1.Purify mouse spleen T cellsHarvest T cells from C57BL/6 mouse spleens using MACS negative selection,and then stained with FTC-CD3e to check the purity.2.The influence of TGP on activation induced T cell deathT cells were plated to 24 well plate,7.5 × 10~5per well,and treated with TGP at the final concentration of 0ug/ml?50 ug/ml?100 ug/ml?200 ug/ml.T cells were stimulated with anti-CD3/CD28.After 24 hours or 48 hours incubation,T cells were haverstedand stained with AnnexinV and 7AAD staining for cell viability.3.The influence of TGP on T cell proliferationT cells were labeled with CFSE firstly,and then plated to 24 well plate,7.5×10~5 per well.T cells were treated with TGP at the final concentration of(0 ug/ml?50 ug/ml?100 ug/ml?200 ug/ml),and stimulated with anti-CD3/CD28.After 48 hours hours incubation,T cells were harvested for flow,we calculated the divisions and percentage of divided T cells.4.The impact of TGP on Fas/FasL mRNA expressionCD4+T cells were harvested and plated in 24 well plate,thentreatedwith TGP atthe concentration of 0ug/ml or 100ug/ml.T cells were stimulated with anti-CD3/CD28.After 24 hours incubation,T cells were lysed for purification of total RNA and synthesis cDNA.Fas or FasL mRNA expression were detected by RT-PCR.5.The impact of TGP on Fas/FasL expressionPurified total T cells were plated to 24 well plate,7.5 x 10~5 per well.T cells were treated with TGP at the concentration of 0ug/mll?100 ug/ml?200 ug/ml.T cells were stimulated withanti-CD3/CD28.After 24 hours or 48 hours incubation,T cells were harvested and stained withFas and FasL,and then tested by flow cytometry.6.The effect of TGP on the expression of apoptosis related protein Bcl-2Purified CD4+ T cells were plated to 24 well plate,7.5 × 10~5 per well.T cells were treated with TGP at the concentration of 0 ug/ml?100 ug/ml?200 ug/ml.T cells were stimulated with anti-CD3/CD28.After 24 hours incubation,T cells were harvested and western blot was used to detect the expression of Bcl-2.Results:1.TGP accelerates activation induced cell deathOur flow data shown that:compared with untreated group,the percentage of live T cells in high dose group decreased significantly at 24 hours.And we also observed significantly decreased percentage of live T cells in middle and high dose TGP groups at 48 hours.2.TGP curbs T cells proliferationCompared with untreated group,all treated gropus have less percentage of divided T cells and divisions.We found TGP can curb T cells proliferation in a concentration depended way,and a high dose of TGP can stop T cells proliferation completely.3.TGP accelerates T cell activation induced cell death by increasing Fas expressionWe found the expression of Fas mRNA was increased significantly after treated with TGP by RT-PCR.Then,we verified Fas expression on the surface of activated T cell,and found the expression of Fas in TGP treated group was increased.4.TGP curbs expression of Bcl-2 in activated T cellsWestern blot data shown that:TGP treated groups have decreased expression of Bcl-2.The higher concentration of TGP,the lower expression of Bcl-2.Conclusion1.TGP curbs mouse T cells proliferation and accelerates activation induced cell death in vitro.2.TGP accelerates activation induced T cell death by increasing Fas expressionand and downregulating Bcl-2 expression.